ORIGINAL ARTICLE Extracellular Signal Regulated Kinase in ...€¦ · Extracellular Signal–Regulated Kinase in the Ventromedial Hypothalamus Mediates Leptin-Induced Glucose Uptake

Extracellular Signal–Regulated Kinase in theVentromedial Hypothalamus Mediates Leptin-InducedGlucose Uptake in Red-Type Skeletal MuscleChitoku Toda,

1Tetsuya Shiuchi,

2Haruaki Kageyama,

3,4Shiki Okamoto,

1,5Eulalia A. Coutinho,

1,5

Tatsuya Sato,1,5

Yuko Okamatsu-Ogura,6Shigefumi Yokota,

1,5Kazuyo Takagi,

1,5Lijun Tang,

1,5

Kumiko Saito,1Seiji Shioda,

3and Yasuhiko Minokoshi

1,5

Leptin is a key regulator of glucose metabolism in mammals, butthe mechanisms of its action have remained elusive. We nowshow that signaling by extracellular signal–regulated kinase (ERK)and its upstream kinase MEK in the ventromedial hypothalamus(VMH) mediates the leptin-induced increase in glucose utilizationas well as its insulin sensitivity in the whole body and in red-typeskeletal muscle of mice through activation of the melanocortin re-ceptor (MCR) in the VMH. In contrast, activation of signal transducerand activator of transcription 3 (STAT3), but not the MEK-ERKpathway, in the VMH by leptin enhances the insulin-induced sup-pression of endogenous glucose production in an MCR-independentmanner, with this effect of leptin occurring only in the presenceof an increased plasma concentration of insulin. Given that lep-tin requires 6 h to increase muscle glucose uptake, the transientactivation of the MEK-ERK pathway in the VMH by leptin mayplay a role in the induction of synaptic plasticity in the VMH,resulting in the enhancement of MCR signaling in the nucleusand leading to an increase in insulin sensitivity in red-type muscle.Diabetes 62:2295–2307, 2013

Leptin is an adipocyte-derived hormone that playsan important role in glucose metabolism in pe-ripheral tissues as well as in overall energy me-tabolism in mammals (1,2). Treatment with leptin

ameliorates diabetes in lipodystrophic mice and humans(3–5) as well as type 1 (6,7) and obesity-unrelated type 2diabetes (8) in rodents. Although the antidiabetic effects ofleptin are known to be mediated by the central nervoussystem (9–11), the mechanism by which leptin stimulatesglucose utilization in muscle has remained unclear.

Neurons in the arcuate hypothalamic nucleus (ARC) andventromedial hypothalamus (VMH) contribute to the effectsof leptin on glucose metabolism. Restoration of expressionof the Ob-Rb receptor for leptin in proopiomelanocortin

(POMC) neurons of db/db mice (which lack Ob-Rb) nor-malizes blood glucose concentration (12,13). The hyper-insulinemia and insulin resistance characteristic of theseanimals remain unaffected, however, suggesting that otherbrain regions may also regulate glucose metabolism. Wepreviously showed that injection of leptin into the VMHincreases glucose uptake by skeletal muscle (mainly the redtype), brown adipose tissue (BAT), and the heart, but not bywhite adipose tissue, through activation of the melanocortinreceptor (MCR) in the VMH (14). These effects of leptin weremanifest at 6 h after injection (14) and were abolished byattenuation of sympathetic nerve signaling through surgicaldenervation or through administration of either a blocker ofsympathetic nerve activity (guanethidine) or the b-adrenergicantagonist propranolol (11,15). Furthermore, whereas lep-tin injection into the VMH increased glucose uptake inmuscle, BAT, and the heart, injection into the ARC increasedglucose uptake in BAT alone, and injection into the dorso-medial hypothalamus (DMH) or paraventricular hypothala-mus (PVH) had no effect (14). The effect of leptin on muscleglucose uptake is thus dependent on Ob-Rb activation in theVMH, as well as on Ob-Rb activation in the ARC.

Activation of Ob-Rb stimulates intracellular signalingpathways, including those mediated by signal transducerand activator of transcription 3 (STAT3), phosphoinositide3-kinase (PI3K), and extracellular signal–regulated kinase 1or 2 (ERK1/2) (1,2,16). Leptin also downregulates the ac-tivity of AMP-activated protein kinase in the ARC and PVH,an effect that contributes to the anorexic action of leptin(17). With the use of a hyperinsulinemic-euglycemic clampand measurement of 2-deoxyglucose (2DG) uptake, wehave now examined the role of leptin signaling in the VMHin the acute effects of leptin injected into the periphery orthe VMH on glucose metabolism in skeletal muscle of leanmice. Our results reveal that signaling by ERK and its up-stream kinase MEK in the VMH mediates the leptin-inducedincrease in glucose utilization and its insulin sensitivity bothin the whole body and in red-type skeletal muscle throughactivation of MCR in the VMH. In contrast, leptin in theVMH was found to enhance the insulin-induced suppressionof endogenous glucose production (EGP), which largelyreflects hepatic glucose production, through a STAT3-dependent, MCR-independent pathway in this nucleus.

RESEARCH DESIGN AND METHODS

Animals. Male FVB mice (CLEA Japan, Tokyo, Japan) were studied at 12–16weeks of age. The animals were housed individually in plastic cages at 246 1°Cwith lights on from 0600 to 1800 h, and they were maintained with free accessto a laboratory diet (Oriental Yeast, Tokyo, Japan) and water. All animalexperiments were approved by the ethics committee for animal experimentsof the National Institute for Physiological Sciences.

From the 1Division of Endocrinology and Metabolism, Department of Devel-opmental Physiology, National Institute for Physiological Sciences, Okazaki,Aichi, Japan; the 2Department of Integrative Physiology, Institute of HealthBiosciences, University of Tokushima Graduate School, Tokushima, Japan;the 3Department of Anatomy, Showa University School of Medicine,Shinagawa-ku, Tokyo, Japan; the 4Faculty of Health Care, Kiryu University,Midori, Gunma, Japan; the 5Department of Physiological Sciences, GraduateUniversity for Advanced Studies (Sokendai), Hayama, Kanagawa, Japan; andthe 6Department of Biomedical Sciences, Graduate School of VeterinaryMedicine, Hokkaido University, Sapporo, Japan.

Corresponding author: Yasuhiko Minokoshi, [email protected] 24 November 2012 and accepted 16 March 2013.DOI: 10.2337/db12-1629This article contains Supplementary Data online at http://diabetes

.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-1629/-/DC1.� 2013 by the American Diabetes Association. Readers may use this article as

long as the work is properly cited, the use is educational and not for profit,and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

diabetes.diabetesjournals.org DIABETES, VOL. 62, JULY 2013 2295

ORIGINAL ARTICLE

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Surgical procedures. A chronic double-walled stainless steel cannula wasimplanted stereotaxically and either unilaterally into the right side of the VMHor bilaterally into the VMH as described previously (14,18). Bilateral cannulaplacement was performed for examination of the effects of bilateral injectionof the MEK inhibitor U0126 or a STAT3 inhibitor into the VMH in those onsystemic injection of leptin. Unilateral cannula implantation was performedfor all other studies. For the hyperinsulinemic-euglycemic clamp, polyethylenecatheters were inserted into the right carotid artery and jugular vein of mice.Animals were handled repeatedly during the recovery period (2 weeks) aftercannula implantation. Correct placement of the cannula tips was verified mi-croscopically in brain sections, with .95% of animals manifesting correctplacement; the few animals with incorrect cannula placement were excludedfrom analysis. Food was removed immediately before the administration ofinhibitors, leptin, or the MCR agonist melanotan-II (MT-II).Administration of leptin, MT-II, and inhibitors. Leptin (5 ng) (PeproTech,Rocky Hill, NJ), the MCR agonist MT-II (10 pmol) (Phoenix Pharmaceuticals,Burlingame, CA), or the MCR antagonist SHU9119 (10 pmol) (Phoenix Phar-maceuticals) in 0.1 mL of physiological saline was injected with a Hamiltonmicrosyringe into the right side of the VMH of freely moving mice through theunilateral cannula. The MEK inhibitor U0126 (10 mmol/L) (Cell SignalingTechnology, Beverly, MA) or the PI3K inhibitor LY294002 (10 mmol/L) (Merck,Darmstadt, Germany) in 0.1 mL of 0.01% DMSO was injected into the same sideof the VMH at 1 h before injection of leptin or MT-II. Cell-permeable SH2domain–binding phosphopeptide (STAT3 inhibitor, Merck) (0.1 mL of a 250 mmol/Lsolution in saline) was injected into the VMH twice, at 1 h and 5 min beforeleptin injection. In some experiments, 0.1 mL of U0126 (10 mmol/L in 0.01%DMSO) or the STAT3 inhibitor (250 mmol/L in saline) was injected into theVMH bilaterally at 1 h or at both 1 h and 5 min, respectively, before in-traperitoneal injection of leptin (5 mg/kg). Control mice were injected with thesame volume of saline or 0.01% DMSO into the VMH or with intraperitonealsaline as appropriate.Hyperinsulinemic-euglycemic clamp and measurement of associ-

ated 2-[14C]DG uptake. Four hours after leptin or MT-II injection, the

hyperinsulinemic-euglycemic clamp protocol was initiated in conscious andunrestrainedmice. The protocol was modified slightly from that described on thewebsite of the Mouse Metabolic Phenotyping Center at Vanderbilt University(http://www.mc.vanderbilt.edu/root/vumc.php?site=mmpc&doc=32773). The120-min basal period (t = –120 to 0 min) was initiated at 1300 h and was fol-lowed by a 105-min clamp period (t = 0–105 min) beginning at 1500 h (Fig. 1A).A priming dose of [3-3H]glucose (5 mCi) (American Radiolabeled Chemicals,St. Louis, MO) was administered via the jugular vein catheter at t = –120 min andwas followed by infusion of the tracer at a rate of 0.05 mCi/min for 2 h. Theclamp period was initiated at t = 0 min by primed and continuous infusion ofbovine insulin (bolus of 16 mU/kg followed by a rate of 5 mU $ kg–1 $ min–1)(Sigma-Aldrich Japan, Tokyo, Japan) through the jugular vein catheter. The rateof [3-3H]glucose infusion was increased to 0.1 mCi/min for the remainder of theexperiment in order to minimize changes in specific activity relative to theequilibration period. Blood was collected every 5–10 min from the carotid arterycatheter, and blood glucose was monitored (One Touch Ultra; Lifescan, Johnson& Johnson). Glucose (30%) was infused at a variable rate via the jugular veincatheter in order to maintain blood glucose levels at 130–150 mg/dL. Withdrawnerythrocytes were suspended in sterile 0.9% saline and returned to each animal.

Tissue 2DG uptake was measured as described previously (14). For as-sessment of 2DG uptake during the basal period, mice were infused with2-[14C]DG (5 mCi) (American Radiolabeled Chemicals) at t = –45 min throughthe jugular vein catheter. At t = –40, –30, –20, –10, and 0 min, an arterial bloodsample (50 mL) was collected for assessment both of the rate of blood glucoseappearance (Ra), which reflects EGP, and of 2DG uptake. For measurement of2DG uptake during the clamp period, another group of mice was infused with2-[14C]DG (5 mCi) at t = 60 min, and blood samples (50 mL) were collected att = 65, 75, 85, 95, and 105 min. Immediately after collection of the final bloodsample (t = 0 or 105 min), mice were killed with an overdose of pentobarbitalsodium, and the soleus, red (Gastro-R) and white (Gastro-W) portions of thegastrocnemius, epididymal white adipose tissue (epiWAT), and liver wererapidly dissected. Gastro-R was dissected from the inner surface of the gas-trocnemius attached to the soleus, whereas Gastro-W was dissected from theouter surface of the muscle. The rate of disappearance of blood glucose (Rd),which reflects whole-body glucose utilization, as well as Ra, the rates ofwhole-body glycolysis and glycogen synthesis, and the rates of glycolysis andglycogen synthesis in muscle were determined as described previously(19,20). Rd is equal to Ra plus the glucose infusion rate (GIR) during the clampperiod, whereas Rd is equal to Ra during the basal period. Plasma concen-trations of insulin (Insulin ELISA; Shibayagi, Gunma, Japan) and glucagon(Glucagon EIA kit; Yanaihara Institute, Shizuoka, Japan) were measured withthe use of kits. Plasma epinephrine and norepinephrine concentrations weremeasured by high-performance liquid chromatography as described previously(18). Glycogen phosphorylase a activity in liver was measured as described

previously (21) and was expressed as the ratio of activity in the absence ofAMP to that in the presence of 3 mmol/L AMP.Immunoblot analysis. The right side of the ARC, VMH, or DMH sampled at 30min after leptin injection into the VMH or at 1 h after intraperitoneal injectionof leptin was dissected from a 1-mm-thick sagittal section prepared fromthe midline of the fresh brain and was subjected to immunoblot analysisas described previously (14) (Supplementary Fig. 1). The primary antibodiesincluded those to Tyr705-phosphorylated STAT3 (pSTAT3), Thr202- and Tyr204-phosphorylated p44/42 MAPK (pERK1/2), and Ser473-phosphorylated Akt(pAkt) (all from Cell Signaling Technology); those to Ser549-phosphorylatedsynapsin (Thermo Scientific Pierce, Rockford, IL); and those to total forms ofthese various proteins (Cell Signaling Technology). All antibodies were used ata dilution of 1:1,000.Immunofluorescence analysis of phosphorylated STAT3 and ERK. At 1 hafter intraperitoneal leptin injection, mice were anesthetized and perfusedtranscardially with 4% paraformaldehyde in 0.1 mol/L phosphate buffer. Braintissue was removed, fixed again, and embedded in OCT compound (SakuraFinetechnical, Tokyo, Japan). Phosphorylated forms of STAT3 and ERK in thesame cryosections (thickness of 7 mm) were detected by consecutive incu-bations with rabbit polyclonal antibodies to Tyr705-phosphorylated STAT3(1:100 dilution) (Cell Signaling Technology) and Alexa Fluor 568–labeled goatantibodies to rabbit immunoglobulin G (1:400 dilution) (Life Technologies,Carlsbad, CA), and then with rabbit polyclonal antibodies to Thr202- andTyr204-phosphorylated ERK1/2 (1:500 dilution) (Cell Signaling Technology)and Alexa Fluor 488–labeled goat antibodies to rabbit immunoglobulinG (1:400 dilution) (Life Technologies). Sections were examined with a fluo-rescence microscope (Olympus AX-70) and a laser confocal microscope(Digital Eclipse C1; Nikon). Staining was absent in control sections processedwithout primary antibodies.Extraction of RNA and reverse transcription PCR analysis. Reversetranscription and real-time PCR analysis with Power SYBR Green PCR MasterMix (Applied Biosystems, Foster City, CA) were performed as describedpreviously (14,18). The sequences of PCR primers (forward and reverse,respectively) were 59-CATGGGCGCAGCAGGTGTATACT-39 and 59-CAAG-GTAGATCCGGGACAGACAG-39 for glucose-6-phosphatase (G6Pase), 59-GGTGTTTACTGGGAAGGCATC-39 and 59-CAATAATGGGGCACTGGCTG-39for PEPCK, and 59-AACTTTGGCATTGTGGAAGG-39 and 59-ACACATTGGGGGTAGGAACA-39 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).Data were normalized by the corresponding abundance of GAPDH mRNA.Statistical analysis. Data are presented as means 6 SEM. Statistical com-parisons between two groups and among multiple groups were performedwith Student t test and with ANOVA followed by Tukey HSD post hoc test,respectively. A P value of ,0.05 was considered statistically significant.

RESULTS

Leptin injection into the VMH stimulates whole-bodyglucose metabolism. We examined the effects of leptininjection into the VMH on glucose metabolism with the useof a hyperinsulinemic-euglycemic clamp (Fig. 1A). Thedose of leptin was selected on the basis of the results ofour previous study (14). We also selected the dose of in-sulin as 5 mU $ kg–1 $ min–1 for the clamp on the basis ofthe results of preliminary experiments showing that thisdose increased the rate of glucose disappearance (Rd,reflecting whole-body glucose utilization) about twofoldand suppressed the rate of glucose appearance (Ra, re-flecting EGP) by about one-half. Immunoblot and immuno-histochemical analyses revealed that leptin injection into theVMH increased the phosphorylation of STAT3, ERK1/2, andAkt (which functions downstream of PI3K) in this brain re-gion but not in the ARC or DMH (Fig. 1B and SupplementaryFig. 2). Preliminary data revealed that the phosphorylationof ERK1/2 peaked at 30 min and returned to the control levelat 6 h after leptin injection into the VMH (data not shown).

During the clamp period, the plasma insulin concentra-tion increased 1.78- and 1.63-fold in mice injected withsaline or leptin, respectively, with these values not differ-ing significantly (Supplementary Table 1). The blood glu-cose concentration was maintained constant by glucoseinfusion (Fig. 1C). Leptin injection into the VMH necessi-tated an increase in GIR (Fig. 1D). The increase in GIR was

LEPTIN IN THE VMH REGULATES GLUCOSE METABOLISM

2296 DIABETES, VOL. 62, JULY 2013 diabetes.diabetesjournals.org

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FIG. 1. Effects of leptin injection into the VMH on whole-body and muscle glucose metabolism in mice during a hyperinsulinemic-euglycemicclamp. A: Experimental protocol for the hyperinsulinemic-euglycemic clamp. B: Phosphorylation of STAT3 (on Tyr

705), ERK (Thr

202/Tyr

204),

and Akt (Ser473

) in the VMH at 30 min after the unilateral injection of leptin (5 ng in 0.1 mL) or saline (0.1 mL) into the VMH. The data wereevaluated with the ratio of phosphorylated form to the total protein and expressed as percent increase of the ratio to that of the saline-injectedgroup. Representative immunoblots with antibodies to the phosphorylated (p) or total (t) forms of each protein are shown above densitometricquantitation of the relative phosphorylated/total protein ratio. *P < 0.05 vs. corresponding value for saline-injected group. C: Blood glucose levelsduring the basal and clamp periods. The clamp period begins at time 0. D: GIR required to maintain euglycemia during the clamp period. *P < 0.05vs. corresponding value for saline-injected group. E: Rate of glucose disappearance (Rd) during the basal and clamp periods in mice injected withleptin or saline into the VMH unilaterally. F: Rate of glucose appearance (Ra) during the basal and clamp periods as well as the percentagesuppression of Ra induced by insulin infusion. Ra reflects EGP. G: Rates of whole-body glycolysis and glycogen synthesis during the clamp period.

C. TODA AND ASSOCIATES


associated with an increase in Rd (Fig. 1E) and a decreasein Ra (Fig. 1F). Leptin injection also significantly increasedwhole-body glycolysis and tended to increase glycogensynthesis (Fig. 1G). Leptin enhanced the insulin-inducedincrease in 2DG uptake in muscle, with this effect beingmore pronounced in red-type muscle (soleus and Gastro-R) than in white-type muscle (Gastro-W) (Fig. 1H). Theincrease in 2DG uptake in red-type muscle was accompa-nied by an increase in glycolysis and glycogen synthesis inthe muscle tissue (Fig. 1I).

In the basal condition, leptin injection into the VMH in-creased Rd (Fig. 1E) without affecting the blood glucoselevel (Fig. 1C). This effect was accompanied by an in-crease in Ra (Fig. 1F), given that Ra is equal to Rd in thesteady-state condition. Leptin increased 2DG uptake insoleus and to a lesser extent in Gastro-R, but not in Gastro-W, similar to its effects during the clamp period (Fig. 1H).Plasma insulin levels did not differ between control andleptin-injected groups during the basal period (Supple-mentary Table 1). Leptin injection into the VMH thus in-creased glucose turnover at the whole-body level duringthe basal period. These results suggested that leptin in-jection into the VMH increases whole-body and muscleglucose utilization under both basal and clamp conditions,whereas it suppresses hepatic EGP in a manner dependenton plasma insulin concentration.Leptin in the VMH induces glucose utilization andenhances insulin-induced suppression of EGPdifferentially via MEK-ERK and STAT3 pathways.We examined the effects of injection of a MEK inhibitor(U0126), a STAT3 inhibitor (cell-permeable SH2 domain–binding phosphopeptide), and a PI3K inhibitor (LY294002)into the VMH. Whereas determination of the acute effectsof specific inhibitors minimizes the influence of adaptiveresponses of neuronal circuits in the brain, the specificityof such agents depends on their concentration. We there-fore first determined the doses of inhibitors that prefer-entially attenuated the activation of their target molecules(Fig. 2A). Plasma insulin and blood glucose levels were notaffected by injection of these inhibitors into the VMH, orby similar injection of leptin, the MCR agonist MT-II, or theMCR antagonist SHU9119 (Supplementary Fig. 3 andSupplementary Table 1). The MEK inhibitor U0126 and theSTAT3 inhibitor, but not the PI3K inhibitor LY294002,suppressed the leptin-induced increase in GIR during theclamp period, with the effect of U0126 being greater thanthat of the STAT3 inhibitor (Fig. 2B). None of these threeinhibitors affected GIR in response to insulin infusionalone. U0126 inhibited the effect of leptin on Rd but not onRa during the clamp period, whereas the STAT3 inhibitorattenuated the effect of leptin on Ra but not on Rd (Fig. 2Cand D). LY294002 had no effect on either Rd or Ra. Theleptin-induced increase in whole-body glycolysis wassuppressed by U0126 but not by the other inhibitors (Fig.2E). Furthermore, the MEK inhibitor, but not the STAT3inhibitor or PI3K inhibitor, attenuated leptin-induced 2DGuptake, glycolysis, and glycogen synthesis in soleus andGastro-R, but not in Gastro-W, during the clamp period(Fig. 3A and C–E). Uptake of 2DG by epiWAT was notaffected by leptin or by the inhibitors (Fig. 3B and F).

U0126 also suppressed the increase in Rd (equal to that inRa) induced by leptin during the basal period (data notshown).

Insulin downregulated the amounts of PEPCK andglucose-6-phosphatase (G6Pase) mRNAs in the liver dur-ing the clamp period, whereas leptin and the leptin sig-naling inhibitors did not affect the hepatic abundance ofthese mRNAs (Supplementary Fig. 4A). In contrast, leptinattenuated the activity of glycogen phosphorylase a in theliver during the clamp period, and this effect was blockedby the STAT3 inhibitor but not by the MEK inhibitor or thePI3K inhibitor (Fig. 3G). These results suggested that lep-tin in the VMH enhances the insulin-induced suppressionof hepatic glucose production via STAT3 signaling in theVMH.

The plasma glucagon concentration did not differ be-tween control and leptin-injected mice either with orwithout insulin infusion (Supplementary Fig. 4B). Injectionof inhibitors of leptin signaling pathways into the VMH alsohad no effect on the plasma glucagon level. Furthermore,plasma norepinephrine and epinephrine concentrationshad decreased to undetectable levels at the end of theclamp period in both control and leptin-injected mice (datanot shown), probably as a result of adequate training andhandling of the animals as well as the high plasma insulinconcentration. Whereas humoral factors may contribute tothe leptin-induced suppression of EGP and hepatic glyco-gen phosphorylase a activity, the effect of leptin on EGPmay be mediated by the autonomic nervous system, in-cluding the vagus nerve, as described previously (8,22,23).Systemic leptin increases glucose utilization viaMEK-ERK signaling in the VMH. The intraperitonealinjection of leptin (5 mg/kg) increased the amounts ofpERK1/2 and pSTAT3, but not pAkt, in the VMH (Fig. 4A).Leptin also increased the amounts of pERK1/2 and pSTAT3in the ARC as well as pSTAT3 in the DMH (SupplementaryFig. 5A). The intraperitoneal injection of leptin increasedthe number of cells positive for both pSTAT3 and pERK inthe VMH (Fig. 4B) as well as in the ARC (SupplementaryFig. 5B). Systemic leptin thus activates ERK and STAT3 inthe same neurons in the VMH as well as in the ARC,probably in a manner dependent on Ob-Rb. Prior bilateralinjection of the MEK inhibitor U0126 into the VMH sup-pressed the increase in ERK phosphorylation in the VMHinduced by intraperitoneal injection of leptin without af-fecting the phosphorylation of STAT3 or Akt in the VMH(Fig. 4A) or of any of the three signaling molecules in theARC or DMH (Supplementary Fig. 5A).

Injection of U0126 into the VMH partially inhibited theincrease in GIR induced by intraperitoneal injection ofleptin (Fig. 4C). This effect of U0126 was associated withsuppression of the leptin-induced increases in Rd (Fig. 4D)and 2DG uptake in the soleus (Fig. 4E). The MEK inhibitordid not affect the leptin-induced enhancement of the sup-pression of Ra by insulin (Fig. 4F). In the basal period,U0126 injection into the VMH inhibited the leptin-inducedincreases in Rd (Fig. 4D), 2DG uptake in the soleus (Fig.4E), and Ra (Fig. 4F), the latter being equal to the increasein Rd (Fig. 4D). Blood glucose and plasma insulin levelsduring the basal or clamp periods were not affected by

H: 2DG uptake in muscle during the basal and clamp periods. I: Rates of glycolysis and glycogen synthesis in muscle during the clamp period. *P <0.05 vs. basal, saline in VMH. †P < 0.05 vs. basal, leptin in VMH. #P < 0.05 vs. clamp, saline in VMH (D–I). All quantitative data are means 6 SEM(n = 6 or 7 mice).














FIG. 2. Leptin injection into the VMH increases whole-body insulin sensitivity via MEK-ERK and STAT3 signaling in the VMH. A: Phosphory-lation of ERK (Thr

202/Tyr

204), STAT3 (Tyr

705), and Akt (Ser

473) in the VMH at 30 min after unilateral injection of leptin or saline into the VMH.

The MEK inhibitor U0126 or PI3K inhibitor LY294002 was injected unilaterally into the VMH at 1 h before leptin injection, whereas the STAT3inhibitor was injected into the VMH at both 1 h and 5 min before leptin injection. DMSO at 0.1% was injected as a control for the inhibitors. Thedata were evaluated with the ratio of phosphorylated form to the total protein and expressed as percent increase of the ratio to that of the saline-injected group. Representative immunoblots with antibodies to the phosphorylated (p) or total (t) forms of the proteins are shown above thequantitative data, which are means 6 SEM (n = 6 or 7 mice). *P < 0.05 vs. corresponding saline-injected group. †P < 0.05 vs. corresponding valuefor leptin + DMSO in VMH. B–E: Effects of leptin and leptin signaling inhibitors on the time course of the increase in GIR (B), on Rd during theclamp period (C), on Ra during the clamp period and on the percentage suppression of Ra induced by insulin infusion (D), and on the rate of whole-body glycolysis or glycogen synthesis during the clamp period (E) for mice subjected to the hyperinsulinemic-euglycemic clamp protocol. The mean



intraperitoneal injection of leptin with or without priorinjection of U0126 into the VMH (Supplementary Fig. 3 andSupplementary Table 1). The enhancement by leptin of theinsulin-induced suppression of hepatic EGP was only par-tially inhibited by injection of the STAT3 inhibitor into theVMH (data not shown). Hepatic EGP is thus regulated byleptin via other brain sites or peripheral tissues as well asvia the VMH.MCR in the VMH mediates leptin-induced glucoseutilization (but not suppression of EGP) in a mannerindependent of MEK-ERK signaling. Injection of theMCR antagonist SHU9119 into the VMH resulted in partialinhibition of the increase in GIR induced by leptin in-jection into the VMH (Fig. 5A). This inhibition of GIR bySHU9119 was associated with suppression of the leptin-induced increases in Rd (Fig. 5B) and 2DG uptake insoleus muscle (Fig. 5C). SHU9119 did not affect the en-hancement by leptin of the insulin-induced suppression ofRa (Fig. 5D). During the basal period, SHU9119 injectioninto the VMH inhibited the leptin-induced increases in Rd(Fig. 5B), 2DG uptake in soleus (Fig. 5C), and Ra (Fig. 5D),with the latter being equal to the increase in Rd (Fig. 5B).

Injection of the MCR agonist MT-II into the VMH in-creased both GIR (Fig. 6A) and Rd (Fig. 6B) during theclamp period. Injection of MT-II into the VMH also enhanced

insulin-induced 2DG uptake in soleus but not in Gastro-W orepiWAT (Fig. 6C). However, the MT-II–induced increases inGIR, Rd, and 2DG uptake in soleus were not inhibited byinjection of the MEK inhibitor U0126 into the VMH (Fig. 6A–C). Moreover, MT-II did not enhance the suppression of Rainduced by insulin during the clamp period (Fig. 6D). Con-sistent with our previous results showing that MT-II in-creased 2DG uptake in red-type skeletal muscle (14), MT-IIincreased both Rd (Fig. 6B) and Ra (Fig. 6D) in the basalperiod, and these effects were not inhibited by U0126.Whereas MT-II did not increase glucose uptake in Gastro-Wduring the clamp period, we previously found that MT-IIinduced a small (1.3-fold) increase in 2DG uptake in whitemuscle in the absence of insulin infusion (14). The effect ofMT-II as well as leptin on glucose uptake and its insulinsensitivity in white skeletal muscle is thus markedly smallerthan in red muscle. These data thus suggested that theincreases in whole-body glucose utilization as well as inglucose utilization by red-type muscle induced by leptin inthe VMH, but not the enhancement by leptin of the sup-pression of EGP by insulin, are mediated by MCR in theVMH.

Leptin has previously been shown to increase the den-sity of hippocampal synapses and of N-methyl-D-aspartate–sensitive glutamate receptors at these synapses in a manner

FIG. 2. (Continued) of the GIR values from 60 to 105 min is shown in the bar graphs (B). All data are means 6 SEM (n = 6 mice). *P < 0.05 vs.corresponding value for saline-injected group. †P< 0.05 vs. corresponding value for leptin + DMSO in VMH. ‡P< 0.05 for leptin + MEK inhibitor inVMH vs. leptin + STAT3 inhibitor in VMH.





FIG. 3. Leptin injection into the VMH increases glucose utilization in red-type muscle via MEK-ERK signaling in the VMH, whereas it enhancesinsulin-induced suppression of glycogen phosphorylase a activity in the liver via STAT3 in the VMH. Effects of leptin and the MEK inhibitor U0126on 2DG uptake (A and B) as well as on glycolysis and glycogen synthesis (C) in soleus, Gastro-R, Gastro-W, or epiWAT during the clamp period ofthe hyperinsulinemic-euglycemic clamp protocol. Effects of leptin, the STAT3 inhibitor, and the PI3K inhibitor LY294002 on 2DG uptake in soleus(D), Gastro-W (E), or epiWAT (F) during the clamp period. G: Effects of leptin and leptin signaling inhibitors on glycogen phosphorylase a activityin liver during the clamp period. All tissue samples were obtained from the mice studied in Fig. 2B–E. All data are means 6 SEM (n = 6 mice). *P<0.05 vs. corresponding value for saline-injected group. †P < 0.05 vs. corresponding value for leptin + DMSO in VMH.



FIG. 4. Systemic injection of leptin increases whole-body glucose utilization and glucose uptake in soleus muscle via MEK-ERK signaling in theVMH. A: Phosphorylation of STAT3 (on Tyr

705), ERK (Thr

202/Tyr

204), and Akt (Ser

473) in the VMH at 1 h after intraperitoneal injection of

leptin (5 mg/kg) or saline (leptin i.p. or saline i.p.). The MEK inhibitor U0126 or DMSO (0.01%) was injected into the VMH bilaterally 1 h beforeinjection of leptin or saline. The data were evaluated with the ratio of phosphorylated form to the total protein and expressed as percent increaseof the ratio to that of saline i.p. + DMSO group. Representative immunoblots with antibodies to the phosphorylated (p) or total (t) forms of theproteins are shown above the quantitative data (n = 6 or 7 mice). *P < 0.05 vs. corresponding value for saline i.p. + DMSO in VMH. §P < 0.05 vs.corresponding value for saline i.p. + MEK inhibitor in VMH. †P < 0.05 vs. leptin i.p. + DMSO in VMH. B: Immunohistofluorescence analysis ofphosphorylated forms of STAT3 (Tyr

705) and ERK (Thr

202/Tyr

204) in the VMH at 1 h after intraperitoneal injection of saline (c, e, and g) or leptin

(a, d, f, and h). Panels d, f, and h are digital zoom images corresponding to the boxed area in panel a, whereas panels c, e, and g represent the



dependent on ERK signaling (24). Finally, we examined theeffects of leptin on the phosphorylation of synapsin, whichcontributes to synapse formation, in the VMH. Leptin in-jection into the VMH increased the phosphorylation ofsynapsin in the VMH in a manner dependent on MEK-ERKsignaling (Fig. 6E).

DISCUSSION

Central or peripheral administration of leptin has beneficialeffects on diabetes in lipodystrophic mice and humans (3–5) as well as on type 1 (6,7) and obesity-unrelated type 2diabetes in rodents (8). We have now shown that MEK-ERKsignaling in the VMH mediates the leptin-induced acute in-crease in glucose uptake in red-type muscle as well as theinsulin sensitivity of this process, and that it thereby con-tributes to the leptin-induced increase in whole-body glu-cose utilization (Fig. 7). In contrast, leptin in the VMHenhances insulin-induced suppression of hepatic EGPthrough the action of STAT3 in the VMH and the inhibitionof glycogen phosphorylase a activity in the liver (Fig. 7).Given that leptin stimulates hepatic EGP under basal con-ditions, it reciprocally regulates this process in a mannerdependent on the plasma insulin concentration. MCR acti-vation in the VMH contributes to the leptin-induced increasein whole-body and muscle glucose utilization but not to itssuppression of EGP. Activation of the MEK-ERK pathway inthe VMH appears to occur upstream of and to be necessaryfor the activation of MCR signaling in the VMH and is re-quired for the increase in muscle glucose uptake.

We previously showed that injection of leptin into theVMH increased muscle glucose uptake in mice at 6 h butnot at 3 h after the injection, whereas intracerebroventricularinjection of the MCR agonist MT-II increased muscle glucoseuptake within 3 h (14). Similarly, intravenous or intra-cerebroventricular injection of leptin was found to inducea slow but progressive increase in sympathetic nerve activityin peripheral tissues (25,26), whereas intracerebroventricularinjection of a-melanocyte–stimulating hormone (a-MSH) re-sulted in immediate activation of sympathetic nerves (26).We previously showed that the increase in muscle glu-cose uptake induced by leptin injection into the VMHis mediated by the sympathetic nervous system andb-adrenergic receptors (b-ARs) (15). We also found thatinjection of the hypothalamic neuropeptide orexin intothe VMH stimulates glucose uptake in the muscle of mice,similar to the effect of leptin, and that this action oforexin is mediated via sympathetic nerves and b2-ARs(18). The orexin-induced increase in glucose uptake wasblunted in b-AR–deficient mice (b-less mice), whereasrestoration of b2-AR expression in the muscle of thesemice resulted in recovery of the orexin effect. Further-more, preliminary data revealed that the phosphorylationof ERK1/2 peaked at 30 min and returned to the controllevel at 6 h after leptin injection into the VMH (data notshown). These observations suggest that leptin exerts itseffects on glucose metabolism by altering neuronal plas-ticity in the VMH through the activation of MEK-ERKsignaling in this region. Consistent with this notion, wefound that leptin injection into the VMH increased the

FIG. 4. (Continued) equivalent area in the VMH. Immunofluorescence of pSTAT3 (c and d), pERK (e and f), and both phosphorylated proteins (a, g,and h) is shown. The dashed trace in panels a and b represents the VMH. Panel b shows a Nissl-stained section of the medial hypothalamus froma C57BL/6 mouse and is modified with permission from Hof et al. (40). 3V, third ventricle. Scale bars, 20 mm. C–F: Effects of injection of the MEKinhibitor U0126 into the VMH on changes in glucose metabolism induced by intraperitoneal injection of leptin in mice subjected to the hyperinsulinemic-euglycemic clamp protocol. U0126 or DMSO was injected into the VMH bilaterally 1 h prior to leptin or saline injection (n = 5 or 6 mice). C: GIR duringthe clamp period. *P < 0.05 vs. corresponding value for saline i.p. + DMSO in VMH. †P < 0.05 vs. corresponding value for leptin i.p. + DMSO in VMH. D:Rd in basal and clamp periods. E: 2DG uptake in soleus, Gastro-W, and epiWAT during the basal and clamp periods. F: Ra during the basal and clampperiods as well as the percentage suppression of Ra induced by insulin infusion. The Ra in the basal period is equal to Rd in the basal period. *P< 0.05 vs.corresponding value for saline i.p. + DMSO in VMH in the basal period. #P < 0.05 vs. corresponding value for saline i.p. + DMSO in VMH in the clampperiod. §P < 0.05 vs. corresponding value for saline i.p. + MEK inhibitor in VMH in the clamp period. ‡P < 0.05 vs. corresponding value for the basalperiod. †P < 0.05 vs. corresponding value for leptin i.p. + DMSO in VMH (D–F). All quantitative data are means 6 SEM.



phosphorylation of synapsin, which contributes to synapseformation, in the VMH in a manner dependent on MEK-ERKsignaling.

We previously showed that injection of leptin into theVMH induced expression of the transcription factor c-FOSin the ARC as well as in the VMH at 6 h after the injection(14). Intense stimulation of VMH neurons by a photo-activatable caged form of glutamate increased the elec-trical activity of POMC neurons in the ARC (27). Moreover,the ventrolateral region of the VMH and the VMH shellcontain a high number of dendrites that harbor a substantialnumber of axons and boutons immunoreactive for a-MSH,and MT-II and SHU9119 reciprocally regulate the activity ofVMH neurons (28). Brain-derived neurotrophic factor isexpressed in the ventrolateral region of the VMH and actsdownstream of MCR in the VMH (29). Brain-derived neu-rotrophic factor enhances neuronal plasticity through ret-rograde action. Early activation of the MEK-ERK signalingpathway in the VMH by leptin may thus induce synapticplasticity in the VMH, resulting in the enhancement of MCR

signaling in the VMH via POMC neurons in the ARC andleading to an increase in insulin sensitivity in red-typemuscle (Fig. 7).

STAT3 and the leptin receptor Ob-Rb in VMH neuronsare implicated in the homeostatic regulation of glucosemetabolism by leptin. Ablation of leptin receptors in ste-roidogenic factor 1 (SF1)–expressing VMH neurons of miceinduced insulin resistance before the onset of obesity (30).Conversely, SF1-specific ablation of suppressor of cytokinesignaling-3 (SOCS-3), a feedback inhibitor of the leptin-induced JAK-STAT3 signaling pathway, improved glucosehomeostasis in mice (31). Furthermore, intracerebroventricularinjection of a STAT3 inhibitor suppressed leptin-inducedenhancement of insulin sensitivity in the liver, and abol-ishment of Ob-Rb–dependent STAT3 signaling (in s/smice)results in pronounced hepatic insulin resistance (32). Ourdata suggest that STAT3 in the VMH regulates liver insulinsensitivity. However, other brain regions, such as the ARCand brain stem, may also contribute to such regulation,given that the enhancement of insulin-induced suppression

FIG. 5. MCR in the VMH mediates leptin-induced whole-body glucose utilization and glucose uptake in soleus muscle, but not leptin-inducedenhancement of the suppressive effect of insulin on EGP. The effects of the MCR antagonist SHU9119 injected into the VMH on changes in glucosemetabolism induced by unilateral injection of leptin into the VMH were evaluated with the hyperinsulinemic-euglycemic clamp. SHU9119 or salinewas injected into the VMH 1 h prior to leptin or saline injection. A: GIR during the clamp period. *P < 0.05 vs. corresponding value for saline +saline in VMH. †P< 0.05 vs. corresponding value for leptin + saline in VMH. B: Rd during basal and clamp periods. C: 2DG uptake in soleus, Gastro-W, and epiWAT during basal and clamp periods. D: Ra during basal and clamp periods as well as the percentage suppression of Ra by insulin in-fusion. Ra in the basal period is equal to Rd in the basal period. *P < 0.05 vs. corresponding value for saline + saline in VMH in the basal period.#P< 0.05 vs. corresponding value for saline + saline in VMH in the clamp period. §P< 0.05 vs. corresponding value for saline + SHU9119 in VMH inthe clamp period. ‡P < 0.05 vs. corresponding value for the basal period. †P < 0.05 vs. corresponding value for leptin + saline in VMH (B–D). Alldata are means 6 SEM (n = 6 or 7 mice).



FIG. 6. An MCR agonist in the VMH increases whole-body glucose utilization and glucose uptake in soleus muscle but does not enhance thesuppressive effect of insulin on EGP. The effects of injection of the MCR agonist MT-II into the VMH on glucose metabolism were evaluated withthe hyperinsulinemic-euglycemic clamp. The MEK inhibitor U0126 or DMSO (0.01%) was injected into the VMH unilaterally at 1 h before injectionof MT-II or saline. A: GIR during the clamp period. *P < 0.05 vs. corresponding value for saline + DMSO in VMH. B: Rd during the basal and clampperiods. C: 2DG uptake in soleus, Gastro-W, and epiWAT during the clamp period. D: Ra during the basal and clamp periods as well as the per-centage suppression of Ra induced by insulin infusion. Ra in the basal period is equal to Rd in the basal period. *P < 0.05 vs. corresponding valuefor saline + DMSO in VMH in the basal period. #P < 0.05 vs. corresponding value for saline + DMSO in VMH in the clamp period. §P < 0.05 vs.corresponding value for saline + MEK inhibitor in VMH in the clamp period. ‡P < 0.05 vs. corresponding value for the basal period (B and D).E: Phosphorylation of synapsin in the VMH after injection of leptin and the MEK inhibitor U0126. The MEK inhibitor U0126 or DMSO (0.01%) wasinjected into the VMH unilaterally at 1 h before injection of leptin or saline without the hyperinsulinemic-euglycemic clamp. The VMH was col-lected at 30 min after leptin injection. The data were evaluated with the ratio of phosphorylated form (p) to the total (t) protein and expressed aspercent increase of the ratio to that of saline + DMSO in the VMH group. *P< 0.05 vs. corresponding value for saline + DMSO in VMH. †P< 0.05 vs.corresponding value for leptin + DMSO in VMH. ns, not significant. All data are means 6 SEM (n = 6 or 7 mice).



of hepatic EGP elicited by intraperitoneal injection ofleptin was only partially attenuated by injection of theSTAT3 inhibitor in the VMH.

We found that leptin suppressed EGP during the clampperiod of the hyperinsulinemic-euglycemic clamp protocol,whereas it increased EGP during the basal period. Theinsulin-dependent reciprocal regulation of EGP by leptinmay explain why leptin does not induce hypoglycemiain the postprandial state. Insulin inhibits hepatic EGP viathe central nervous system (33) as well as through the in-hibition of agouti-related peptide–containing neurons in theARC (34). Insulin in the brain modulates leptin signaling inthe hypothalamus (35). The increased plasma insulin levelsduring the prandial state may thus influence the effect ofleptin on hepatic EGP in both the brain and liver.

PI3K signaling in the hypothalamus also regulates glu-cose and lipid, as well as energy, metabolism (36–38).Ablation of the p110a catalytic subunit of PI3K in POMCneurons was found to impair glucose metabolism (36). Incontrast, ablation of this subunit in SF1-expressing neu-rons did not affect glucose homeostasis (37). Theseobservations as well as our present data suggest that thePI3K pathway in Ob-Rb–expressing neurons in other brainregions, such as the ARC, rather than in the VMH, mightcontribute to the effects of leptin on glucose metabolism.Furthermore, the PI3K pathway in Ob-Rb–expressingneurons in the VMH might contribute to the chronic ratherthan the acute effects of leptin on glucose metabolism.

We previously showed that whereas leptin injection intothe VMH increased glucose uptake in muscle, BAT, and theheart, injection into the ARC increased glucose uptake inBAT but not in muscle or heart, and injection into the DMHor PVH had no effect (14). Injection of MT-II either into theVMH or intracerebroventricularly increased glucose uptake

in muscle, BAT, and the heart, whereas injection into thePVH increased glucose uptake in BAT alone, and injectioninto the DMH or ARC had no effect (14). Thus, whereasleptin-induced glucose uptake in BAT and muscle is medi-ated by Ob-Rb and MCR in the VMH, glucose uptake in BATmay also be mediated by Ob-Rb in POMC neurons in theARC and then MCR in the PVH. PVH neurons project to thesolitary nucleus and to the raphe nucleus, and therebystimulate thermogenesis in BAT (39). Further investigationis necessary to explore the neuronal pathway responsiblefor leptin-induced glucose uptake in muscle from the VMHto the hindbrain and muscle tissue.

In summary, we have found that the MEK-ERK pathwayin the VMH plays an important role in leptin-induced glu-cose uptake in red-type muscle as well as whole-bodyglucose utilization. MCR in the VMH also mediates theseeffects of leptin. In contrast, activation of STAT3 in theVMH by leptin enhances the insulin-induced suppressionof hepatic EGP by inhibiting glycogen phosphorylase aactivity. These results suggest that VMH neurons mediatethe antidiabetic effects of leptin in the control of muscle aswell as liver glucose metabolism. They thus provide im-portant insight into the mechanism of the antidiabeticeffects of leptin in humans as well as rodents.

ACKNOWLEDGMENTS

This work was supported by Grants-in-Aid for ScientificResearch (B) (21390067 and 24390058 to Y.M.), Grants-in-Aid for Young Scientists (B) (20790656 and 22790875 toS.O. and 23790282 to C.T.), and a Grant-in-Aid for ScientificResearch on Innovative Areas (Research in a ProposedResearch Area, “Molecular Basis and Disorders of Controlof Appetite and Fat Accumulation;” 22126005 to Y.M.) from

FIG. 7. Model for the mechanism of regulation of glucose metabolism in muscle and liver by leptin in the VMH. Ob-Rb in the VMH plays a key role inthe regulation of glucose metabolism and insulin sensitivity in muscle and liver by leptin. Leptin-activated MEK-ERK signaling in the VMHincreases insulin sensitivity and glucose utilization in red muscle through activation of MCR in the VMH. Ob-Rb–expressing VMH neurons likelyactivate POMC neurons either in the ARC itself (14) or at their synaptic connections with VMH neurons through the MEK-ERK pathway. The MEK-ERK pathway then stimulates synaptic plasticity for POMC neurons and MCR-expressing neurons in the VMH. Whereas other brain sites maycontribute to the leptin-induced enhancement of the suppressive effect of insulin on hepatic glucose production, leptin-activated STAT3 signalingin the VMH mediates this enhancement by inhibiting glycogen phosphorylase a activity in liver. 3V, third ventricle.



the Ministry of Education, Culture, Sports, Science andTechnology of Japan, as well as by the Specific ResearchFund of the National Institutes for Natural Sciences (toY.M.).

No potential conflicts of interest relevant to this articlewere reported.

C.T. researched data, contributed to discussion, and wroteand edited the manuscript. T.S., H.K., and S.S. researcheddata, contributed to discussion, and reviewed and edited themanuscript. S.O. contributed to discussion. E.A.C., T.S.,Y.O.-O., S.Y., K.T., L.T., and K.S. researched data. Y.M.designed the study, contributed to discussion, and wroteand edited the manuscript. Y.M. is the guarantor of thiswork and, as such, had full access to all the data in the studyand takes responsibility for the integrity of the data and theaccuracy of the data analysis.

The authors thank N. Kawai and K. Nagatani for labora-tory management, the Center for Analytical Instruments atthe National Institutes for Basic Biology (Okazaki, Japan)for biochemical analysis, and K.W. Brocklehurst, an in-dependent scientific editorial consultant (Washington, DC),for editorial assistance.

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